S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Ph.D. / Sc.D._농생명공학부)
Functional characterization of gene knockout mutants of Autographa californica multiple nucleopolyhedrovirus
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- Autographa californica multiple nucleopolyhedrovirus ; ac11 ; ac43 ; ac78 ; BV production ; ODV envelopment ; virus morphology
- 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2013. 8. 제연호.
- The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which has a large double-stranded DNA genome of approximately 134 kb and comprises 154 open reading frames (ORFs), highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. The function of AcMNPV genes in viral replication has been studied using gene knock-out technology, however, the function of more than one-third of viral genes which including some highly conserved genes are still unknown. In this study, to investigate the function of AcMNPV genes, a novel AcMNPV genome that can be maintained in Escherichia coli as a plasmid and infect susceptible lepidopteran insect cells was generated. This engineered AcMNPV, named Ac-MK, contains an E. coli origin of replication (mini-F replicon) and a kanamycin resistance gene (Kan). Using a convenient Tn7 transposon-based plasmid capture system, pPCS-S, which contains a pUC origin and an ampicillin resistance gene (Amp), 54 single ORF-knockout AcMNPV mutants were generated by random insertion into Ac-MK genome. Subsequently, the growth properties of these ORF-knockout mutant viruses in Spodoptera frugiperda 9 (Sf9) cells, and the gene knockout-specific effects on the production of infectious progeny were analyzed. Three of these mutants, Ac11KO, Ac43KO and Ac78KO, of which ORF11 (ac11), ORF43 (ac43), and ORF78 (ac78) were knocked-out respectively, were selected and subjected to further study since ac11, ac43 and ac78 are highly conserved genes in baculovirus which suggest that they may play important roles in the baculovirus life cycle.
The result of quantitative PCR (qPCR) analysis revealed that ac11 is an early gene in the viral life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in the Ac11KO-transfected Sf9 cells. However, qPCR analysis demonstrated that deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid observed in cytoplasm or plasma membrane in Ac11KO-transfected cells, which demonstrated that the defection of BV production from the Ac11KO-transfected cells was due to inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived virus (ODV), and their subsequent embedding into the occlusion bodies (OBs) was also blocked in the Ac11KO-transfected cells demonstrated that ac11 is required for ODV envelopment. These results therefore demonstrated that ac11 is an essential gene in the viral life cycle.
The function of ac43 gene during viral replication was also investigated. After transfection into Sf9 cells, Ac43KO produced polyhedra much larger in size than those of wild-type AcMNPV. Interestingly, some of the nucleocapsids were singly enveloped in the polyhedrin matrix while the nucleocapsids of AcMNPV are known to be multiply enveloped. Furthermore, Ac43KO led to a defect in the transcription and expression of polyhedrin, which resulted in reduced OB production. However, Ac43KO did not affect production of BV as there was no remarkable difference in budded virus titer. These results suggest that ac43 plays an important role in the expression of polyhedrin, the morphogenesis of OB, and the assembly of virions occluded in OBs.
Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. In the transfected Sf9 cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious BVs were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and ODVs revealed that Ac78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that Ac78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrated that Ac78 is a late virion-associated protein and is essential for the viral life cycle.